Membrane tension and peripheral protein density mediate membrane shape transitions

Abstract

Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins.

Figure 1. Endophilin N-BAR domain induced membrane tubulation of GUV

(a) Sketch of a micro-pipette aspirated GUV. ΔP is the pressure difference between inside and outside of the pipette used for GUV aspiration. R_p and R_v represent the pipette radius and the radius of the spherical part of the GUV, respectively, L_p represents the aspiration length of the GUV. (b) The process of transferring an aspirated GUV from the GUV dispersion (red) into a protein solution (green) (also see Methods). (c) Time lapse confocal images showing the formation of tubes (after t=24s, as indicated by arrows) and the change in aspiration length during endophilin N-BAR binding. Membrane tension was held constant at 0.05 mN·m⁻¹. Green: protein channel; Red: lipid channel. (d) ① A GUV incubated to equilibrium with endophilin N-BAR under high tension (0.25 mN·m⁻¹). After equilibration, tension was reduced to 0.07 mN·m⁻¹ within 2 seconds. Membrane tubes as indicated by arrows can be observed on the GUV under low membrane tension (0.07 mN·m⁻¹) for Δt equal to ② 8s; ③ 20s; ④ 48s (Δt=0 is defined as the time point of tension reduction). Scale bars: 10 µm.

Figure 2. Membrane tension and bound protein density modulate membrane shape transition

(a) A representative trial with high (0.206 mN·m⁻¹) membrane tension, the membrane-bound endophilin N-BAR density at the onset of area decrease (as indicated by the arrow) genuinely reveals the shape transition point. The area is calculated from the time-dependent aspiration length and vesicle radius as shown in Supplementary Figure 2b. (b) A representative trial with low (0.029 mN·m⁻¹) membrane tension. Transition-density (marked by the dashed lines) decreased significantly compared to the high tension case shown in (a). Bulk concentrations of endophilin N-BAR are 150nM in (a) and 75nM in (b). Potential influence of bulk protein concentration on transition-densities was eliminated by comparing the transition-densities of similar tension GUVs in endophilin N-BAR solutions of various bulk concentrations (Supplementary Figure 5a). Additionally, there was no observable influence of membrane tension on the endophilin N-BAR’s equilibrium density on GUVs (Supplementary Figure 5b).

Figure 3. Experimental shape stability diagram agrees well with curvature instability theory

Filled triangles represent the measured transition-density (expressed as a cover fraction, using the close-packed N-BAR dimer density of 30000 µm⁻²) of GUVs under corresponding tensions. The open data points represent the maximum protein cover fraction reached by a GUV with (triangle) or without (circle) tubulation during protein-membrane binding. The solid line represents the best fit of experimental data with the proposed curvature instability model (r²=0.85). The dashed lines are 95% confidence intervals for the fit. The shaded area represents the region where the membrane is tubulated by endophilin N-BAR. The arrows indicate two ways of inducing membrane tubulation: 1), by increasing protein coverage on the membrane at constant tension or 2), by decreasing membrane tension at constant coverage. The large circle (non-tubulated state), and triangle (tubulated state), represent the state of the membrane before and after tension reduction (compare Figure 1d), respectively. The inset shows the same data using linear axes. Error bars represent the standard errors associated with determining each data point. Concentrations of endophilin N-BAR used in the experiment ranged from 25nM to 400nM (also note Supplementary Figure 5a).

Figure 4. Full length protein shows smaller curvature generation capacity than N-BAR

Transition-densities of full length endophilin (blue triangles) as well as the best fit with our curvature instability model (blue line, r²=0.75) are plotted on top of the stability diagram of N-BAR displayed in Figure 3 for GUVs with the same lipid composition (DOPS/DOPE/DOPC = 45/30/25). The physical properties resulting from fitting the endophilin full length data are: the spontaneous curvature C₀⁻¹ = 6.1±1.1nm; the upper tension limit σ* = 0.17±0.04 mN·m⁻¹; the protein transition-density required for tubulating a tensionless membrane ρ₀ = 850±300µm⁻². P=0.035 between the stability boundaries of endophilin full length and N-BAR via f-test. Error bars represent the standard errors associated with determining each data point.

Figure 5. Membrane charge affects equilibrium density, not transition density

Equilibrium densities of endophilin N-BAR (open bars) increase significantly for increasing amounts of DOPS in the GUV (for each composition pair P<10⁻⁴, Student t test). No significant difference can be found among the transition densities (gray bars, for each composition pair, P>0.5, Student t test). Concentration of endophilin N-BAR domain: 100nM. GUV compositions: DOPS/DOPE/DOPC = X/30/(70-X). All GUVs considered here are at the membrane tension of 0.095 ± 0.013 mN·m⁻¹ (Mean ± SD). Gray error bars are standard deviations (SD) of the data and black error bars are standard errors of the mean (SEM), same below.

Figure 6. The effect of conical lipids on membrane shape transitions

Under the same membrane tension (0.068 ± 0.007 mN·m⁻¹ (Mean ± SD))), the presence of 30% conical lipids, either DOPE or cholesterol, significantly lowers the transition-density of endophilin N-BAR domain. ***P<10⁻⁴, Student t test.

Figure 7. Three ways of mediating membrane curvature instability

Three regulatory elements are identified in this contribution that can modulate membrane shape transitions induced by the binding of curvature coupling proteins. Notably, membrane budding and tubulation is not solely induced by protein association (left arrow). The effects of lowering membrane tension (middle arrow) and changing membrane lipid composition (right arrow) also control membrane shape transition without the assistance of additional proteins. The contribution of peripheral proteins is defined by their density on the membrane, emphasizing a thermodynamic role played by protein molecules in mediating membrane shape transitions. The tension effect may explain an ultrafast pathway cells can utilize to control membrane shape transformations such as endocytosis.